The present invention relates to radiolabeled compounds and their use in radioimaging and/or radiotherapy. More particularly, the present invention relates to radiolabeled irreversible inhibitors of epidermal growth factor receptor tyrosine kinase (EGFR-TK) and their use as biomarkers for medicinal radioimaging such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), and as radiopharmaceuticals for radiotherapy.
The use of radioactive nuclides for medicinal purposes is well known in the art. Biologically active compounds that bind to specific cell surface receptors or that in other ways modify cellular functions has received some consideration as radiopharmaceuticals, and therefore, when labeled with a radioactive nuclide, such compounds are used as biospecific agents in radioimaging and radiotherapy.
Positron Emission Tomography (PET), a nuclear medicine imagine technology which allows the three-dimensional, quantitative determination of the distribution of radioactivity within the human body, is becoming an increasingly important tool for the measurement of physiological, biochemical, and pharmacological function at a molecular level, both in healthy and pathological states. PET requires the administration to a subject of a molecule labeled with a positron-emitting nuclide (radiotracer) such as 15O, 13N, 11C and 18F, which have half-lives of 2, 10, 20, and 110 minutes, respectively.
Single Photon Emission Computed Tomography (SPECT) is a form of chemical imaging in which emissions from radioactive compounds, labeled with gamma-emitting radionuclides, are used to create cross-sectional images of radioactivity distribution in vivo. SPECT requires the administration to a subject of a molecule labeled with a gamma-emitting nuclide such as 99mTc, 67Ga, 111In and 123I.
Polypeptides such as growth factors, differentiation factors, and hormones often mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic intracellular protein tyrosine kinase activity. Epidermal growth factor receptor-tyrosine kinase (EGFR-TK) is over expressed in breast cancer and other neoplasia. A suitable radiotracer that binds to EGFR-TK might allow, through a nuclear medicine imaging technique such as PET and SPECT, the mapping and quantification of this receptor-kinase. This would allow the study of changes in levels of expression of this receptor, including the monitoring of response to hormonal or other chemotherapy, and could lead to better patient management and differentiation in regard to therapeutic course of action.
Moreover, such radiotracer that comprises a suitable radioactive nuclide can be further used as an EGFR-TK biospecific agent for radiotherapy.
Recently, 99mTc-labeled anti EGFR antibody was synthesized and biodistribution and dosimetry studies were performed in humans [1, 2]. However this labeled antibody, similar to other protein radiopharmaceuticals, has high and prolonged retention of radioactivity in the liver which constitutes a major problem for clinical applications. Furthermore, the researchers found that it was difficult to obtain accurate quantification of activity in tumors within normal organs because of varying background activities, particularly in lung lesions where fluid and atelectasis could not be differentiated from tumor.
EGF itself has been labeled for nuclear medicine imaging with gamma emitting nuclides including 99mTc [3, 4] and indium-111 [5, 6], and the positron-emitting nuclide bromine-76 [7, 8]. The biodistribution in normal rats of the latter, bromine-76 EGF (murine), was reported [8], but no other in vivo studies in laboratory animals or humans have been reported.
4-Anilinoquinazolines, also referred to herein as 4-(phenylamino)quinazolines, have been shown to potently and selectively inhibit EGFR-TK activity by binding reversibly to an inner membrane ATP binding site on EGFR-TK, the prototype for such compounds being the small-molecules PD 153035 [9] and AG 1478 [10]. A report of a radioiodinated analog of PD 153035 including in vitro binding studies in MDA-486 cells has been presented [11]. PD 153035 labeled with carbon-11 in the 6,7-methoxy groups has been evaluated in rats implanted with human neuroblastoma xenografts (SH-SY5Y) but specific uptake was not determined in a blocking study [12]. PD 153035 was also labeled with carbon-11 specifically in the 7-methoxy position and biodistribution experiments were performed in normal mice, but uptake specificity could not be demonstrated as administration of an enzyme-blocking dose of PD 153035 caused an increase in tracer uptake in the tissues studied [13]. The same abstract reported the labeling of the 7-(2-fluoroethoxy) PD 153035 analog with fluorine-18, but no biological experiments with this tracer were described. Additionally, the 2-18F-fluoroethyl group might be subject to a high rate of 18F-hydrogen fluoride elimination to give the corresponding alkene ether, potentially resulting in high uptake of fluorine-18 in bone, giving poor in vivo images. Further, these ultra potent (IC50 less than 30 pM) inhibitors may only measure flow or permeability surface area rather than biochemical changes [14].
U.S. Pat. No. 6,126,917 teaches 4-(anilino)quinazoline derivatives, reversible inhibitors of EGFR-TK, labeled with fluorine-18 on the aniline ring. These compounds were tested in vitro, in vivo and by PET image analysis. While some of these compounds showed effective (reversible) inhibition activity in vitro, they were found to be ineffective as tracers for the imaging of EGFR-TK in vivo due to kinetic factors such as kon and koff and rapid blood clearance, as was further demonstrated by an animal PET comparative study between fluorine-18 FDG and these radiolabeled compounds. It is assumed that the discrepancy between the encouraging in vitro results and the discouraging in vivo results derives from the ATP competition at the compounds"" binding site.
Thus, in order to achieve better imaging results, the non-specific binding of the radiolabeled compounds should be reduced. This can potentially be achieved by the use of derivatives of irreversible EGFR-TK inhibitors that are labeled with a positron-emitting nuclide. The irreversible binding of such compounds could potentially result in higher diagnostic performance. Furthermore, such irreversible inhibitors, when labeled with a suitable radioactive nuclide, can be used as effective radiotherapy agents as well, based on their high affinity toward, and irreversible binding to, tumor cells expressing EGFR-TK. Thus, such radiolabeled compounds that are targeted to the EGF receptor can bind preferentially to tumor cells and would lead to a high effective concentration of the radionuclides and therefore cause preferential cell killing at the site of the tumor.
Irreversible EGFR-TK inhibitors were recently described [15, 16 and U.S. Pat. Nos. 6,153,617 and 6,127,374]. The irreversible binding thereof is achieved by 4-(anilino)quinazoline derivatives that are substituted at the 6 or 7 position of the quinazoline ring with an xcex1,xcex2-unsaturated carboxylic group, preferably an acrylamide group, which binds covalently to the Cys-773 at the EGFR-TK ATP binding site. Some of these compounds showed high potency toward EGFR inhibition in both in vitro and in vivo experiments. However, these compounds were not radiolabeled, and therefore cannot be used for radioimaging or radiotherapy.
There is thus a widely recognized need for, and it would be highly advantageous to have, radiolabeled irreversible inhibitors of EGFR-TK for use in radioimaging and radiotherapy.
According to the present invention there are provided novel radiolabeled compounds that are irreversible inhibitors of EGFR-TK and methods of using same in radioimaging and radiotherapy.
Thus, according to one aspect of the present invention there is provided a radiolabeled compound of a formula: 
Wherein:
Q1 is Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino, or Q1 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino and Q2 is Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z;
X is selected from the group consisting of xe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94NR1xe2x80x94, NHxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94NHxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94CH2xe2x80x94 and xe2x80x94CHR1xe2x80x94Sxe2x80x94 or absent;
Y is selected from the group consisting of a non-radioactive carbon and a radioactive carbon;
Z is selected from the group consisting of xe2x80x94R2Cxe2x95x90CHR3, xe2x80x94Cxe2x89xa1Cxe2x80x94R3 and xe2x80x94R2Cxe2x95x90Cxe2x95x90CHR3;
Ra is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;
A, B, C and D are each independently selected from the group consisting of hydrogen, a non-radioactive derivatizing group and a radioactive derivatizing group selected from a radioactive bromine, a radioactive iodine and a radioactive fluorine;
R1 is selected from the group consisting of hydrogen and substituted or non-substituted alkyl having 1-6 carbon atoms;
R2 is selected from the group consisting of hydrogen, halogen and alkyl having 1-6 carbon atoms; and
R3 is selected from the group consisting of hydrogen, halogen, carboxy, alkenyl, alkoxy carbonyl, substituted or non-substituted alkyl having 1-6 carbon atoms and substituted or non-substituted phenyl;
provided that the compound comprises at least one radioactive atom.
According to further features in preferred embodiments of the invention described below, the non-radioactive derivatizing group is selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, hydroxy, alkoxy, carboxy, carbalkoxy, thiohydroxy, thiocarboxy, thioalkoxy, alkylsulfinyl, alkylsulfonyl, amino, diamino, carbamyl, dicarbamoyl, nitro and cyano.
According to still further features in the described preferred embodiments Q1 is Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z and Q2 is selected from the group consisting of hydrogen, halogen, alkoxy, hydroxy, thiohydroxy, thioalkoxy, alkylamino and amino.
According to still further features in the described preferred embodiments Q1 is Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z and Q2 is hydrogen.
According to still further features in the described preferred embodiments X is xe2x80x94NR1xe2x80x94 and Z is xe2x80x94R2Cxe2x95x90CHR3.
According to still further features in the described preferred embodiments each of R1, R2 and R3 is hydrogen.
According to still further features in the described preferred embodiments Y is a radioactive carbon.
According to still further features in the described preferred embodiments at least one of A, B, C and D is a radioactive fluorine.
According to still further features in the described preferred embodiments D is a radioactive fluorine.
According to still further features in the described preferred embodiments D is a radioactive fluorine, A and B are each chlorine and C is hydrogen.
According to still further features in the described preferred embodiments A is a radioactive bromine or a radioactive iodine.
According to still further features in the described preferred embodiments the radioactive carbon is carbon-11.
According to still further features in the described preferred embodiments Y is carbon-11, A and B are each chlorine, C is hydrogen and D is fluorine.
According to still further features in the described preferred embodiments the radioactive fluorine is fluorine-18.
According to still further features in the described preferred embodiments the radioactive bromine is bromine-76 or bromine-77.
According to still further features in the described preferred embodiments the radioactive iodine is iodine-123 or iodine-124.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the radiolabeled compound of the invention and a pharmaceutical acceptable carrier.
According to yet another aspect of the present invention there is provided a method of monitoring the level of epidermal growth factor receptor within a body of a patient comprising (a) administering to the patient the radiolabeled compound of the invention; and (b) employing a nuclear imaging technique for monitoring a distribution of the compound within the body or within a portion thereof.
According to still further features in the described preferred embodiments the technique is positron emission tomography or single photon emission computed tomography.
According to still another aspect of the present invention there is provided a method of radiotherapy comprising administering to a patient a therapeutically effective amount of the radiolabeled compound of the invention.
According to an additional aspect of the present invention there is provided a method of synthesizing a radiolabeled compound of a formula: 
Wherein:
Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z is at position 6 or 7 of the quinazoline ring;
X is selected from the group consisting of xe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94NR1xe2x80x94, NHxe2x80x94CHR1xe2x80x94, CHR1xe2x80x94NHxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94CH2xe2x80x94 and xe2x80x94CHR1xe2x80x94Sxe2x80x94 or absent;
Y is carbon-11;
Z is selected from the group consisting of xe2x80x94R2Cxe2x95x90CHR3, xe2x80x94Cxe2x89xa1Cxe2x80x94R3 and xe2x80x94R2Cxe2x95x90Cxe2x95x90CHR3;
Ra is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;
A, B, C and D are each independently selected from the group consisting of hydrogen and a non-radioactive derivatizing group;
R1 is selected from the group consisting of hydrogen, and substituted or non-substituted alkyl having 1-6 carbon atoms;
R2 is selected from the group consisting of hydrogen, halogen and alkyl having 1-6 carbon atoms; and
R3 is selected from the group consisting of hydrogen, halogen, carboxy, alkenyl, alkoxy carbonyl, substituted or non-substituted alkyl having 1-6 carbon atoms and substituted or non-substituted phenyl.
The method comprising: (a) coupling an aniline derivatized by the Ra, A, B, C and D with a 4-chloroquinazoline substituted at position 6 or 7 by a reactive group, so as to produce a reactive 4-(phenylamino)quinazoline derivatized by the Ra, A, B, C and D; and (b) reacting the reactive 4-(phenylamino)quinazoline with a reactive carbon-11 labeled xcex1,xcex2-unsaturated carboxylic derivative.
According to still further features in the described preferred embodiments the Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z group is at position 6 of the quinazoline ring.
According to still further features in the described preferred embodiments the reactive 4-(phenylamino)quinazoline is 4-(phenylamino)-6-nitroquinazoline, and the method further comprising, prior to step (b), reducing the 4-(phenylamino)-6-nitroquinazoline so as to produce a 4-(phenylamino)-6-aminoquinazoline derivatized by the A, B, C and D.
According to still further features in the described preferred embodiments the reactive carbon-11 labeled xcex1,xcex2-unsaturated carboxylic derivative is carbon-11 labeled acryloyl chloride.
According to yet an additional aspect of the present invention there is provided a method of synthesizing a radiolabeled compound of formula II as described hereinabove, wherein:
Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z is at position 6 or 7 of the quinazoline ring;
X is selected from the group consisting of xe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94NR1xe2x80x94, NHxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94NHxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94CH2xe2x80x94 and xe2x80x94CHR1xe2x80x94Sxe2x80x94 or absent;
Y is a non-radioactive carbon;
Z is selected from the group consisting of xe2x80x94R2Cxe2x95x90CHR3, xe2x80x94Cxe2x89xa1Cxe2x80x94R3 and xe2x80x94R2Cxe2x95x90Cxe2x95x90CHR3;
Ra is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;
A, B, C and D are each independently selected from the group consisting of (i) hydrogen, (ii) a non-radioactive derivatizing group and (iii) fluorine-18, provided that at least one of A, B, C and D is fluorine-18;
R1 is selected from the group consisting of hydrogen, and substituted or non-substituted alkyl having 1-6 carbon atoms;
R2 is selected from the group consisting of hydrogen, halogen and alkyl having 1-6 carbon atoms; and
R3 is selected from the group consisting of hydrogen, halogen, carboxy, alkenyl, alkoxy carbonyl, substituted or non-substituted alkyl having 1-6 carbon atoms and substituted or non-substituted phenyl.
The method comprising: (a) preparing a fluorine-18 labeled aniline derivatized by the Ra, A, B, C and D, wherein at least one of A, B, C and D is fluorine-18; (b) coupling the fluorine-18 labeled aniline derivatized by the Ra, A, B, C and D with 4-chloroquinazoline substituted at position 6 or 7 by a reactive group, so as to produce a reactive fluorine-18 labeled 4-(phenylamino)quinazoline derivatized by the A, B, C and D; and (c) reacting the reactive fluorine-18 labeled 4-(phenylamino)quinazoline with a reactive xcex1,xcex2-unsaturated derivative.
According to still further features in the described preferred embodiments the reactive fluorine-18 labeled 4-(phenylamino)-quinazoline is fluorine-18 labeled 4-(phenylamino)-6-nitroquinazoline and the method further comprising, prior to step (c), reducing the fluorine-18 labeled 4-(phenylamino)-6-nitroquinazoline so as to produce a fluorine-18 labeled 4-(phenylamino)-6-aminoquinazoline derivatized by the A, B, C and D.
According to still an additional aspect of the present invention there is provided a method of synthesizing a radiolabeled compound of formula II as described hereinabove, wherein:
Xxe2x80x94Y(xe2x95x90O)xe2x80x94Z is at position 6 or 7 of the quinazoline ring;
X is selected from the group consisting of xe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94NR1xe2x80x94, NHxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94NHxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94CHR1xe2x80x94, xe2x80x94CHR1xe2x80x94CH2xe2x80x94 and xe2x80x94CHR1Sxe2x80x94 or absent;
Y is a non-radioactive carbon;
Z is selected from the group consisting of xe2x80x94R2Cxe2x95x90CHR3, xe2x80x94Cxe2x95x90Cxe2x80x94R3 and xe2x80x94R2Cxe2x95x90Cxe2x95x90CHR3;
Ra is selected from the group consisting of hydrogen or alkyl having 1-8 carbon atoms;
A, B, C and D are each independently selected from the group consisting of (i) hydrogen, (ii) a non-radioactive derivatizing group and (iii) a radioactive atom selected from a radioactive bromine and a radioactive iodine, provided that at least one of A, B, C and D is a radioactive bromine or a radioactive iodine;
R1 is selected from the group consisting of hydrogen, and substituted or non-substituted alkyl having 1-6 carbon atoms;
R2 is selected from the group consisting of hydrogen, halogen and alkyl having 1-6 carbon atoms; and
R3 is selected from the group consisting of hydrogen, halogen, carboxy, alkenyl, alkoxy carbonyl, substituted or non-substituted alkyl having 1-6 carbon atoms and substituted or non-substituted phenyl.
The method comprising: (a) coupling an aniline derivatized by the Ra, A, B, C and D, wherein at least one of A, B, C and D is a halogen atom, with 4-chloroquinazoline substituted at position 6 or 7 by a reactive group, so as to produce a reactive 4-(phenylamino)quinazoline derivatized by the A, B, C and D; (b) radiolabeling the reactive 4-(phenylamino)quinazoline derivatized by the A, B, C and D with a radioactive bromine or a radioactive iodine, so as to produce a radioactive bromine labeled or a radioactive iodine labeled reactive 4-(phenylamino)quinazoline derivatized by the A, B, C and D, wherein at least one of the A, B, C and D is a radioactive bromine or a radioactive iodine; and (c) reacting the radioactive bromine labeled or radioactive iodine labeled reactive 4-(phenylamino)quinazoline with a reactive xcex1,xcex2-unsaturated derivative.
According to still further features in the described preferred embodiments the reactive 4-(phenylamino)-quinazoline is 4-(phenylamino)-6-nitroquinazoline and the method further comprising, prior to step (b), reducing the 4-(phenylamino)-6-nitroquinazoline, so as to produce a 4-(phenylamino)-6-aminoquinazoline derivatized by the A, B, C and D, wherein at least one of the A, B, C and D is a halogen.
According to still further features in the described preferred embodiments the halogen is bromine.
The present invention successfully addresses the shortcomings of the presently known configurations by providing novel irreversible biomarkers for radioimaging and radiopharmaceuticals for radiotherapy.